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跨膜突变型TNF |
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曾劲杨 李卓娅 龚非力 徐 勇 熊 平
摘 要 目的：跨膜型TNF-α(TM-TNF-α)易在某些蛋白酶的作用下转换为分泌型TNF-α(S-TNF-α)。拟用基因突变技术获得稳定表达的、不能转换成S-TNF的TM-TNF突变体(TM-TNFm)。方法：应用RT-PCR技术，从人外周血单核细胞中扩增出编码TM-TNF-α的全长cDNA序列并构建pBSK-TNF-α重组体。以此为模板，借助改进的“一次重组PCR”定位突变技术，造成TM-TNF转换为S-TNF酶切位点的缺失，获得TM-TNF突变重组体(TM-TNFm)。结果：将TM-TNFm cDNA片段克隆至表达载体pGEM-3Zf，利用体外转录翻译系统表达出具有生物学活性的跨膜突变型TNF-α蛋白。经Western blot分析证实，用胶原酶不能将TM-TNF突变体酶解成为S-TNF。结论：提示所构建的TM-TNFm去除了可转换为S-TNF的酶解氨基酸序列，为进一步研究跨膜型TNF杀瘤作用奠定了基础。
关键词 跨膜型TNF-α(TM-TNF-α) 跨膜型TNF-α突变体(TM-TNFm-α) 一次PCR定位突变(S-PCR) 体外转录翻译
中国图书分类号 R392.11
Construction and in vitro translation of transmembrane tumor necrosis factor-α
mutant gene
ZENG Jin-Yang, LI Zhuo-Ya, GONG Fei-Li et al.
Department of Immunology,Tongji Medical University,Wuhan 430030
Abstract Objective：Transmembrane TNF-α (TM-TNF-α) can readily be converted by certain proteinase into secretary TNF-α(S-TNF-α). Therefore, in the present study, the authors attempt to make use of mutagenesis technique for constructing a kind of stable expressed TM-TNF mutant (TM-TNFm) which would not be cleaved into S-TNF. Methods: A full length of TM-TNF-α cDNA was amplified from the human monocytes by RT-PCR and cloned into plasmid pBSK to construct recombinant pBSK-TM-TNF. pBSK-TM-TNF-α mutant was then obtained by deletion of enzymatic site for conversion of TM-TNF into S-TNF-α with the site-directed mutagenic method of modified "Single-Step PCR". Results: The mutant was subcloned into expression plasmid pGM-3Zf and then in vitro transcribed and translated into protein which displayed its biological activities. As shown by Western blot, TM-TNF mutant could not be speciafically hydrolyzed into S-TNF by collagenase. Conclusion:These results suggested that TM-TNFm produced was indeed void of the amino acids sequence that could b[1] [2] [3] 下一页
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